Preparation method

ABSTRACT

A method of performing an immunologic evaluation of a subject, comprising collecting a whole blood sample from the subject, maintaining the sample for at least 6 hours after collection, purifying a population of cells comprising lymphocytes and antigen presenting cells from the maintained sample, optionally by a process incorporating a positive or negative affinity selection step to remove granulocytes, and using the purified cells in a cell-mediated immunoassay (CMI assay).

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation in part of U.S. applicationSer. No. 12/419,068, pending, which is a continuation in part ofInternational Patent Application No. PCT/GB2007/003800, filed Oct. 5,2007, published on Apr. 10, 2008 as International Patent Publication No.WO 2008/041004, which claims priority to Application No. GB 0619853.5,filed Oct. 6, 2006, all of which applications are incorporated herein intheir entireties.

FIELD OF THE INVENTION

The present invention relates to methods for storing and purifying cellsfrom a whole blood sample, e.g., a whole blood sample. In particular,the invention relates to methods for storing and purifying cells fromblood samples, such that a population of lymphocytes and antigenpresenting cells is effectively stabilised and purified for use inassays which measure cell mediated immune responses in vitro.

BACKGROUND TO THE INVENTION

Cell Mediated Immune (CMI) responses are commonly used to define theimmune status of an individual. Typically, in the art of clinicalimmunology, the term CMI response encompasses in vivo skin testing,lymphocyte proliferation assays, and the in vitro detection of cytokinesproduced by peripheral blood mononuclear cells (PBMC) in the presence ofa specific antigen. The invention described herein addresses improvedmethods for manipulating, stabilising and preparing cells derived fromisolated whole blood samples for use in assay techniques designed tomeasure one class of CMI responses, namely the in vitro cytokine-basedCMI response to a specific antigen (hereinafter referred to as a “CMIAssay”).

The cells of the immune system are capable of producing immune effectormolecules such as cytokines following stimulation by an antigen. CMIAssays involve incubating a cell sample with an antigen and measuringfor the presence or quantity of an immune effector molecule such as acytokine to provide an indication of the ability of the individual togenerate a cell-mediated immune response to the selected antigen. Cellsfor use in a CMI Assay also include isolated populations of lymphocytes(particularly T Cells) and Antigen Presenting Cells (APCs). APCs areinvolved in processing the antigen in order that the latter may berecognised by T Cell receptors located on the surface of each T Cell.Antigen-induced cytokines may be released into the assay medium anddetected directly by, for example, ELISA methods, or quantified in termsof the frequency of cytokine-secreting T Cells using ELISPOT methods.

The filter immunoplaque assay, otherwise called the enzyme-linkedimmunospot assay (ELISPOT), was initially developed to detect andquantitate individual antibody-secreting B cells. At the time it wasdeveloped, the technique provided a rapid and versatile alternative toconventional plaque-forming cell assays. Recent modifications haveimproved the sensitivity of the ELISPOT such that cells producing as fewas 100 molecules of a specific protein per second can be detected. Theseassays take advantage of the relatively high concentration of a givenproteinaceous cell product (such as a cytokine) in the environmentimmediately surrounding the protein-secreting cell. These cell productsare captured and detected using high-affinity antibodies. The ELISPOTassay has been reviewed in Current Protocols in Immunology (1994; Pub.John Wiley & Sons, Inc.), Unit 6.19 pages 6.19. 1-8.

The ELISPOT assay typically involves six steps: (1) coating a purifiedcytokine-specific antibody to a membrane-backed microtiter plate; (2)blocking the plate to prevent non-specific absorption of any otherproteins; (3) incubating the cytokine-secreting cells with appropriatereagents; (4) removal of cells and reagents; (5) adding a labelledsecond anti-cytokine antibody; and (6) detecting the antibody-cytokinecomplex on the membrane.

Methods for isolating subsets of immune cells for analysis using ELISPOTassays have previously been disclosed. See Peters et al., Methods inMolecular Biology, Handbook of ELISPOT: Methods and Protocols (2005),302, pp. 95-115. Current methods to prepare cells from a whole bloodsample for use in a CMI assay involve the isolation of peripheral bloodmononuclear cells (PBMCs) using density separation methods such as aFicoll gradient. In accordance with these methods, and in order to beeffective in the CMI assay, lymphocytes and APCs must be purified fromthe whole blood sample as soon as possible, and in particular within 8hours of collection of the blood sample from an individual. Janetzski,S. et al., Chapter 4: “Standardisation and Validation Issues of theELISPOT assay”, in Handbook of ELISPOT (Ed. A. E. Kalyuzhny; HumanaPress, New Jersey; 2005), page 80, Note 5. This latter observation wasconfirmed by Meier et al., who showed that the number of spot-forming Tcells as measured in a CMI assay diminished significantly followingstorage of isolated blood for 1-2 days. Meier et al., Eur. J. Clin.Microbiol. Infect. Dis. (2005), 24, pp. 529-536; see FIG. 2 inparticular.

Several authors have suggested that granulocytes impair T cell function.In 1998, Saxton and Pockley examined the expression of the activationmarker CD11 b on neutrophils (a type of granulocyte) present in wholeblood isolated from healthy laboratory volunteers. Following incubationat Room Temperature (RT) and 4° C., these authors showed that expressionof the CD11b antigen by peripheral blood neutrophils was up-regulatedafter relatively short periods of in vitro incubation. Saxton andPockley, J. of Immunological Methods (1998) 214:11-17 and Bartels andSchoorl, Clin. Lab. Haem (1998) 20, 165-168 also examined granulocyteactivation and degranulation following storage of whole blood isolatedfrom 20 subjects, using CD63 and CD67 granulocyte degranulation markers,and demonstrated an increase in mean CD63 and CD67 antigen expressionfollowing blood storage. Bartels and Schoorl also showed that, followingwhole blood incubation, granulocytes ‘shift’ into the monocyte region ofa blood cell scattergram, as defined by the Sysmex NE-8000 haematologyanalyser used in these studies. Bartels and Schoorl, Clin. Lab. Haem(1998) 20: 165-168.

Other studies have provided evidence suggesting that: (a) whenactivated, granulocytes can be responsible for inhibiting T cellfunction; (b) the release of reactive oxygen species during theactivation of granulocytes contributes to T cell dysfunction; and (c) acrucial T cell-signalling molecule, namely p56^(lck) (a tyrosinekinase), is degraded following exposure of T cells to activatedgranulocytes. Schmielau & Finn Cancer Research (2001) 61: 4765-4760;Malmberg et al., The Journal of Immunology (2001) 167:2595-2601; andCemerski et al., Eur. J. Immunol. (2003) 33:2178-2185. Cemerski et al.summarized the state-of-the-art in 2003 by pointing out (see Cemerski etal., Introduction) that reactive oxygen species were known to affectprotein structure by inducing various side chain modifications oncysteines, tyrosines, methionines, prolines, etc., by forming proteincross-linkages, and by oxidising the protein backbone, resulting inprotein fragmentation. Collectively, these observations indicate that Tcell function in stored blood samples is likely to be impaired whenmeasured in a CMI assay.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows IFNγ ELISpot responses (duplicate) to CEF peptide fromcells isolated by (i) Negative Affinity Selection with CD15 MagneticBeads (shown by a solid line marked with diamonds:

), and (ii) Ficoll separation (shown by a broken line marked withsquares:

) from four different donors (A to D).

SUMMARY OF THE INVENTION

The present inventors have identified methods of maintaining or storinga whole blood sample, then purifying, from the blood sample, apopulation of lymphocytes and APCs for use in a cell-mediatedimmunoassay (CMI assay), such as an ELISPOT assay. Methods are alsoidentified for treating whole blood samples prior to storage. Thesemethods allow whole blood samples to be maintained, for example, for atleast 6 hours, such as up to 48 hours before use in a CMI assay, such asan ELISPOT assay.

In accordance with the present invention, there is provided a method ofstoring and purifying cells from a whole blood sample collected from asubject, comprising storing the sample for at least 6 hours aftercollection, then purifying a population of lymphocytes and APCs from thestored sample, in order to remove granulocytes, for use in a CMI assay,by a process incorporating a positive or a negative affinity selectionstep.

In accordance with another aspect of the present invention, there isprovided a method of treating and storing a whole blood sample collectedfrom a subject, comprising treating said whole blood sample by dilutionwith cell growth medium; and storing the treated whole blood sample forat least 6 hours after collection, wherein after storage a population oflymphocytes and APCs can be obtained from the sample for use in a CMIassay. After storage, PBMCs or a suitable preparation of lymphocytes andAPCs can be isolated from the diluted whole blood sample for use in aCMI assay, such as an ELISPOT assay.

In another aspect of the present invention, there is provided a methodof ‘rescuing’ or maintaining the antigen- (or peptide) specific cytokineresponse of isolated T cells derived from a whole blood sample in anELISPOT assay, comprising separating a population of cells comprising Tcells and APCs from said whole blood sample using a positive or negativeaffinity selection.

In a further aspect of the present invention, there is provided a methodof preparing lymphocytes and APCs suitable for use in an ELISPOT assay,wherein said method comprises removing red blood cells by filtration.

In some embodiments, the whole blood sample collected from a subject andthe cells purified from the whole blood sample are not subject toconditions sufficient to effect differentiation of precursor effector Tcells (or memory T cells) in the sample or the purified cells toimmediate effector T cells before or during a CMI assay (e.g., anELISPOT assay). Accordingly, detection of antigen-specific cytokineproduction in the CMI assay using these purified cells indicates thepresence of antigen-specific immediate effector T cells in the subject.

Thus, in a preferred non-limiting embodiment, the present inventionprovides for a method of detecting antigen-specific immediate effector Tcells in a subject, comprising (a) collecting a whole blood sample fromthe subject, (b) storing the sample for at least 6 hours after thecollection, wherein storage may optionally include or embody shipment ortransport of the whole blood sample, (c) purifying a population of cellscomprising lymphocytes (e.g., T cells) and antigen presenting cells fromthe stored sample by a process selected from the group consisting ofpositive affinity selection and negative affinity selection in order toremove granulocytes, (d) contacting the purified cells with a surfacecarrying an immobilized antibody to a cytokine, (e) presenting to thepurified cells an activating amount of the antigen in the absence of anyantigen presenting cells pre-cultured with the antigen, (f) incubatingthe purified cells under conditions to permit release of the cytokinebut where the incubation time is not sufficient to effectdifferentiation of precursor effector T cells (or memory T cells) toimmediate effector T cells, and (g) detecting the cytokine released inresponse to the antigen and bound to the immobilized antibody, whereinthe whole blood sample and the purified cells are not subject toconditions sufficient to effect differentiation of precursor effector Tcells (or memory T cells) to immediate effector T cells before theincubation. An advantage of this method is that the interval betweencollection of the blood sample and purifying the lymphocytes and antigenpresenting cells may be greater than 6 hours, or greater than 8 hours,or greater than 12 hours or greater than 24 hours, and up to 48 hours.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of storing and purifying cellsfrom a whole blood sample collected from a subject for use in a CMIassay such as an ELISPOT assay. The present invention also provides amethod of treating a whole blood sample by dilution with cell growthmedium and storing the treated whole blood sample. The method yields asufficient level of peptide-specific cytokine response from purified orisolated lymphocytes (in the presence of APCs) from said blood sample tobe detected in a CMI assay such as an ELISPOT assay, and preferably tobe useful in a diagnostic or monitoring method. The subject may havebeen previously exposed to the peptide (or antigen).

In accordance with the present invention, a cell mediated immunoassay(CMI assay) refers to an in vitro assay to measure the cytokine-basedcell mediated immune response to a specific antigen in which thepresence of cells other than lymphocytes and antigen presenting cells isundesirable. Non-limiting examples of CMI assays includecytotoxic-T-lymphocyte assays, tetramer staining, proliferation assays,intracellular cytokine staining assays, ELISA assays, and ELISPOTassays. Such an assay uses a sample obtained from an individual or asubject, wherein the sample comprises cells of the immune system. Thecells of the immune system are capable of producing immune effectormolecules such as cytokines following stimulation by an antigen. Suchassays include incubating the sample with an antigen and measuring forthe presence or quantity of the immune effector molecules such ascytokines to perform an immunologic evaluation of the subject or toprovide an indication of the ability of the subject to generate a cellmediated immune response to the selected antigen. In accordance with thepresent invention, the CMI assay is preferably an ELISPOT assay. Cellsfor use in the CMI assay such as an ELISPOT assay include isolatedpopulations of lymphocytes and APCs, such as a population of T cells andAPCs.

In a whole blood sample collected from a subject previously exposed to aspecific antigen (for example, a peptide derived from a virus), thereare very few antigen-specific effector T cells (also known as immediateeffector T cells) that are capable of producing cytokines without firstundergoing cell division or differentiation (also known as immediateeffector T cells), and antigen-specific memory T cells that cannotproduce cytokines without differentiation to immediate effector T cells.When re-exposed to the same antigen, these antigen-specific memory Tcells (also known as precursor effector T cells) can divide anddifferentiate to immediate effector T cells.

Particular types of ELISPOT assays have been developed for detectingantigen-specific cytokine responses by immediate effector T cells from asubject previously exposed to the antigen. Typically, these cells havealready been exposed to the specific foreign antigen in vivo, and, whenre-exposed to the same ‘cognate’ antigen ex vivo, their cytokineresponse is triggered very rapidly (e.g., within 6 hours of in vitroexposure of the cells to the antigen)—that is, the antigen-specific Tcell response is not dependent on cell division or differentiation. SuchELISPOT assays have been previously described in, for example, Lalvaniet al., 1997, J. Exp. Med. 186:859-65 and International PatentApplication No. PCT/GB97/03222, published on Jun. 4, 1998 asInternational Patent Publication No. WO 98/23960, and U.S. Pat. No.7,575,870. In these assays, cells isolated from a subject are incubatedwith the antigen under conditions (e.g., an incubation time period of16-20 hours) insufficient to effect differentiation of memory T cells orprecursor effector T cells to immediate effector T cells such that thedetection of antigen-specific cytokine release indicates the presence ofantigen-specific immediate effector T cells in the subject.

Other types of CMI assays include whole blood assays, where, followingspecific antigen stimulation ex vivo, cytokine released from T cellspresent in whole blood is measured in isolated plasma using standardELISA techniques (e.g., Arend et al., 2000, J. Infect. Dis.,181:1850-54).

The isolated preparation of lymphocytes and APCs, in particular T cellsand APCs, prepared from a whole blood sample in accordance with thepresent methods provides a preparation that can be used in a CMI assaysuch as an ELISPOT assay for use in diagnosis or monitoring. Such apreparation generates responses sufficient to allow diagnosis, or forother purposes, for example, monitoring the progress of, or resistanceto, a chronic infectious disease in the subject, and monitoringinduction and maintenance of antigen-specific T cells followingimmunization of the subject with a vaccine. Such responses may bedefined as 50% higher than background. Alternatively, such responses maybe defined as being at least 50% of the maximal response that can beobtained using a freshly isolated whole blood sample.

For example, in a typical assay, 2.5×10⁵ viable PBMCs may be used perwell in an ELISPOT assay. In an ELISPOT assay for tuberculosis,typically, a negative control will have less than 5 spots. In this case,a positive or reactive sample will have 6 or more spots more than thenegative control. If the negative control has 6 or more spots, areactive sample is indicated if it contains more than two times thenegative control spot count.

In one embodiment, the methods of the present invention use a positiveaffinity selection step or a negative affinity selection step to removegranulocyte cells from a whole blood sample, before or after storage. Apositive affinity selection step is used to bind those cells of interestto a solid support and thus separate bound cells from non-bound cells.The purified bound cells are retained, and the non-bound cells arediscarded.

In the alternative, a negative affinity selection step is used in orderto remove those cells which are not required or undesirable in the cellpreparation (e.g., granulocytes) for use in the CMI assay, such as anELISPOT assay. Thus the cells bound to the solid support are discardedand the purified unbound cells are recovered and retained.

The method will result in purification of both lymphocytes (such as Tcells) and APCs. The purification may comprise positive or negativeaffinity selection of both lymphocytes and APCs, in which case therewill typically be an enrichment in the sample of both of these celltypes, i.e. both the ratio of T cells to total number of cells and theratio of APCs to total number of cells will increase in the sample as aresult of carrying out the method of the invention.

In accordance with one aspect of the invention, the whole blood sampleis maintained or stored before purifying cells from the whole bloodsample by a method or process including a positive or negative affinityselection step. For example, the whole blood sample may be stored priorto isolation of the selected cell population. After storage for at least6 hours, or at least 8 hours, up to 10, 12, 18, 24, 36 or 48 hours, thewhole blood sample is treated (i.e. subjected to the affinity selection)to isolate the selected cell population, which can then be used in theCMI assay, such as an ELISPOT assay. In the alternative, the whole bloodsample is treated (i.e., subjected to the affinity selection) shortlyafter it has been obtained, for example, within 1 hour, up to 6 hours,or up to 8 hours after it has been obtained to isolate the selected cellpopulation. Such a cell population is then used in the CMI assay, suchas the ELISPOT assay.

Preferably, the whole blood sample is stored between −5° C. and 40° C.,typically between 2° C. and 8° C. or between 18° C. and 25° C. In aparticularly preferred embodiment, the whole blood sample is stored atroom temperature, such as at about 18° C. or 20° C. or from 18° C. to25° C. or from 20° C. to 25° C. Where cell division and/ordifferentiation (e.g., differentiation of precursor effector T cells ormemory T cells to immediate effector T cells) is not desired, the wholeblood sample may be kept under conditions not sufficient to effect suchdivision and/or differentiation. For example, the whole blood sample maybe maintained or stored for less than 24 hours, 16 hours or 12 hours.

It will be understood that the storage step, during which a whole bloodsample is maintained for a specified combination of time andtemperature, may optionally include or embody the act of physicallyshipping or transporting the blood sample from one geographical locationto another geographical location under conditions in which the said timeand temperature parameters are generally maintained. This storage modemay involve transport by road, rail, or air, and represents an importantaspect of the overall storage element of the invention, since anindividual whose immune status is to be analysed, and from whom a bloodsample has been obtained, is often located at a considerable distancefrom the laboratory which undertakes the analysis, for example, by meansof performing a cell-mediated immunoassay.

Typically, a whole blood sample, housed in a suitable leak-proof vessel,can be shipped inside an insulated container (e.g., a ThermosafeInsulated Shipper—VIP Model # 07VIP-UPS; available from TegrantCorporation, Arlington Heights, Ill. 60004). Optionally, devices whichare capable of controlling temperature variation during shipment towithin a few degrees are placed in close proximity with the bloodsample. For example, the Phase 22™/20-24° C. pouch from CryopakCorporation (available from TCP Reliable, Edison, N.J. 08837) is able tomaintain contained blood samples at between 20-24° C. during shipment inhot or cold external environments. The time periods required forshipment within regions of the USA (from the point of departure of thesample to the place of its receipt) are well within the storage timeperiods falling within the scope of the invention. And it will beunderstood that the shipment time (that is, the period during which asample is shipped from location A to location B) may represent all ofpart of the storage or maintenance period as specified in the invention.

The shipped blood sample is removed from the storage container at thedestination location, then further processed to generate purified cellscontaining lymphocytes and antigen presenting cells in accordance withthe invention.

In a further embodiment the sample is not frozen at any stage of any ofthe methods mentioned herein. In another embodiment the sample is notfrozen between the stages of being taken or collected from theindividual and being stored and purified (by a method of the invention);and/or the purified cells from the sample are not frozen between thestages of being stored and purified and being used in a CMI assay.

In one aspect of the present invention, the purification methodcomprises a negative affinity selection step to remove granulocytes andoptionally red blood cells from the whole blood sample in order toobtain the preparation of lymphocytes and APCs for use in an ELISPOTassay.

Thus, in accordance with the one aspect of the present invention, amethod is provided in which both granulocytes and red blood cells areremoved from the whole blood sample in a single step. In accordance withthis method, the whole blood sample is contacted with an antibodypreparation comprising anti-CD66b and glycophorin A antibodies. Theantibodies serve to aggregate red blood cells and granulocytes. Theaggregated red blood cells and granulocytes can be removed from thesample, for example by centrifugation. The sample may also be subjectedto a Ficoll gradient prior to centrifugation.

In another aspect of the invention, the negative affinity selectioncomprises the use of a solid support having a ligand bound thereto whichbinds to a cell surface protein present on the surface of granulocytes,which does not bind to cell surface proteins present on the surface oflymphocytes and APCs used in the ELISPOT assay. For example, a solidsupport can be provided comprising an anti-CD15 ligand to bind to CD15+cells from the sample and/or anti-CD66b to remove CD66b+ cells from thesample.

In accordance with this aspect of the present invention, a whole bloodsample, or a blood sample which has been treated to remove red bloodcells is brought into contact with the solid support comprising ananti-CD15 ligand or anti-CD66b ligand, under conditions which allowbinding of granulocytes. The preparation of lymphocytes or APCs havinggranulocytes removed therefrom can be obtained directly, for example bycontacting the sample with the solid support and collecting any materialwhich does not bind the solid support.

In the alternative, the solid support having granulocytes or otherunwanted cells bound thereto can be separated from the remainder of thesample. For example, the solid support can comprise beads such asmagnetic beads. Once the beads have been contacted with the sample andunder conditions to allow binding of granulocytes to the solid support,the beads can be separated from the sample to leave a preparation oflymphocytes and APCs having granulocytes removed therefrom. In apreferred embodiment, the solid support comprises magnetic beads whichcan be separated from the remainder of the sample, for example by theapplication of a magnetic field.

It will be understood that other ligands, particularly antibodies,capable of binding to a cell surface marker on the surface ofgranulocytes, may be used for negative affinity selection. For example,the following antibodies may also be used to remove granulocytes by thismethod: anti-CD16b and/or anti-CD88.

Preferably, in accordance with this aspect of the present invention redblood cells are also removed from the whole blood sample. This can bedone before, after or at the same time as removal of CD15+ expressingcells or granulocytes.

Red blood cells can be removed from the sample by any suitable method.For example, red blood cells in the sample may be removed by lysis ofthe red blood cells (Simon et al., Immunol. Commun. 1983, Vol. 12, pp.301-314).

In accordance with one aspect of the invention, red blood cells areremoved by filtration. In accordance with this aspect of the invention,a method is provided for preparation of lymphocytes and APCs for use ina CMI assay such as an ELISPOT assay which comprises filtration toremove red blood cells. Such methods can be used on their own or withone or more of the other methods of the invention, such as negativeaffinity selection with anti-CD15+ ligands to remove granulocytes.

The filtration methods of the present application include applying awhole blood sample to a filter. The filters are selected such that redblood cells pass through the filter while lymphocytes and APCs areretained on the filter. In accordance with this method, the filtershaving lymphocytes and APCs thereon can be used directly in the CMIassay such as the ELISPOT assay. Alternatively, the lymphocytes and APCscan be collected, for example by washing the filter or back flushing thefilter and collecting the recovered lymphocytes and APCs.

In accordance with another aspect of the present invention, apreparation of lymphocytes and APCs are purified from a whole bloodsample by a method involving a positive affinity step, whereinoptionally the affinity selection step selects all types of T cells (forexample irrespective of whether they are CD4 or CD8 or irrespective ofthe epitope which they recognize).

In one aspect, the whole blood sample is contacted with a solid supporthaving attached thereto ligands which bind to cell surface proteinspresent on the surface of the lymphocytes and APCs. The ligands may beantibodies.

In a preferred aspect of the present invention, such ligands areselected from anti-CD4, anti-CD8, anti-CD19, anti-CD45, anti-CD45RC andanti-CD14 ligands. For example, a solid support can be provided to bindto CD4+, CD8+ CD 14+ and CD 19+ cells in the whole blood sample or asubset such as CD4+, CD8+ and CD19+ cells. Isolation of such cellsprovides a preparation of lymphocytes and APCs which are suitable foruse in the ELISPOT assay. Such a preparation may encompass a mixture ofCD4+ and CD8+ T lymphocytes, B cells and monocytes. In one embodiment,anti-CD4 and/or anti-CD8 ligands are not used in the purification step,and preferably anti-CD4 antibodies and/or anti-CD8 antibodies are notused in the purification step.

In a preferred embodiment, the ligands on the solid support are selectedso that undesirable cells in the final preparation, such asgranulocytes, are not retained on the solid support. For example, theligands are selected so that they are specific for lymphocyte and APCsand do not bind to granulocytes. The solid support can be provided withanti-CD4, anti-CD8 and anti-CD19 ligands or other appropriate ligands inapproximately the same ratios. Alternatively, the ratio of eachindividual ligand can be selected, for example to reflect the proportionof each cell present in whole blood. In the alternative, the ratio ofligands, such as CD4:CD8:CD19 ligands can be selected to bind andisolate lymphocytes and APCs in a desired ratio, such as oneparticularly suitable for use in an ELISPOT assay.

Methods of affinity selection using solid supports having ligands boundthereto to bind to selected proteins are well known in the art. Ingeneral, a whole blood sample is brought into contact with the solidsupport comprising the ligands of interest, under conditions which allowbinding of cells to the ligands via the relevant cell surface protein.The solid support can be separated from the remainder of the sample inorder to separate bound cells from non-bound material. Washing steps maybe included, for example to rinse non-bound material from the solidsupport.

In a preferred aspect of the present invention, the solid supportcomprises magnetic beads. Such magnetic beads can easily be separatedfrom a sample, for example by the application of a magnetic field. Wherethe solid support is provided in the form of magnetic beads or othertypes of bead, each bead may have a single ligand present thereon, suchas anti-CD4 ligand. Beads which individually carry each ligand, such asanti-CD4, anti-CD8, anti-CD14 or anti-CD19 can be combined together toproduce a solid support which will bind to the desired cells, such asCD4+, CD8+, CD14+ and CD19+ cells. In the alternative, each magneticbead may be provided with more than one ligand such as both anti-CD4 andanti-CD8 ligands or anti-CD4 and anti-CD19 ligands or a combination ofall ligands. In a particularly preferred embodiment, the ratio ofligands, such as anti-CD4:anti-CD8:anti-CD19 is selected to correlate tothe ratio of lymphocytes and APCs desired in the final preparation foruse in a CMI assay (for example, an ELISPOT assay). The quantity ofmagnetic beads can also be selected in order to isolate a selectednumber of cells, to facilitate further processing of the preparation foruse in the CMI assay.

The preparation of selected cells, and in particular lymphocytes andAPCs that are bound to the solid support can be used in an assaydirectly. In particular, magnetic beads having bound thereto therelevant cells can be used in a CMI assay, such as an ELISPOT assay.Preferably, the beads having cells bound thereto are washed prior to usein the assay. In the alternative, once the selected cells have beenisolated from the remainder of the whole blood sample, the cells boundto the solid support can be dissociated or separated from the solidsupport for use in the assay.

In accordance with another non-limiting embodiment of the presentinvention, which may or may not include an affinity selection step, awhole blood sample is treated by dilution with cell growth medium andstored before being used in the CMI assay such as an ELISPOT assay. Thediluted whole blood sample can be stored for at least 6 hours aftercollection for example for at least 8 hours after collection from anindividual, preferably up to 10, 12, 18, 24, 36 or 48 hours aftercollection. Where cell division and/or differentiation (e.g.,differentiation of precursor effector T cells or memory T cells toimmediate effector T cells) is not desired, the treated whole bloodsample may be kept under conditions not sufficient to effect suchdivision and/or differentiation. For example, the whole blood sample maybe maintained or stored at 2-8° C.

In accordance with this aspect of the present invention, the whole bloodsample is stabilized or treated prior to storage. For example, themethod comprises diluting a whole blood sample with cell growth medium,such that when the whole blood sample or an isolated preparation of Tcells and APCs obtained from said stored whole blood sample is used inan ELISPOT assay up to 48 hours after collection of the sample a viableresponse is maintained.

In accordance with the present invention, the whole blood sample can bediluted with cell growth medium in any appropriate ratio. In a preferredembodiment, the ratio of whole blood sample to cell growth medium is1:1. Alternatively, the ratio of whole blood sample to cell growthmedium can be 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, up to 10:1,20:1 or 30:1. Alternatively, the ratio of whole blood sample to cellgrowth medium can be 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10, upto 1:15, 1:20 or 1:30.

In accordance with one embodiment, the diluting medium is added to thewhole blood sample, but is not subjected to shaking or mechanicalmixing. The whole blood sample is preferably stored in a standardheparinized tube. Preferably, the whole blood sample is stored in thedark.

The cell growth medium or diluting medium for use in the invention canbe any appropriate cell growth medium or isotonic saline solution. Forexample, the medium can be a commercially available medium such as AIM-V(Invitrogen Paisley UK), or RPMI 1640™ (Sigma Aldrich Corp, St. Louis,Mo., USA). In a preferred embodiment, the cell growth medium isserum-free medium, such as AIM-V.

In accordance with one aspect of the invention said treated whole bloodsample is used or prepared for use in a CMI assay after storage.Typically, a preparation of PBMCs, or T lymphocytes and APCs arepurified from said treated whole blood sample after storage for use inthe CMI assay. Any suitable method can be used to obtain the isolatedpreparation of cells for use in a CMI assay, such as an ELISPOT assay.Typically, PBMCs can be isolated, for example, using a Ficoll gradient.More preferably, red blood cells and granulocytes are removed from thetreated whole blood sample, for example to isolate a preparation oflymphocytes and APCs, by a method including an affinity selection step.

Treatment by dilution may also be used in another aspect of theinvention to treat an isolated preparation of lymphocytes and APCs priorto storage of the preparation before its use in a CMI assay.

In a further aspect of the invention, whole blood may be maintained orstored for several hours, preferably for at least 6 hours, or up to 12hours, or from 12-48 hours. Such storage may be at 2-8° C., or at roomtemperature. Following storage, a preparation of lymphocytes and APCsmay be prepared by affinity selection as described above, and optionallythe peptide specific response of T cells in the preparation may bemeasured in a CMI assay. In this manner, the in vitro activity of Tcells introduced into the CMI assay is stabilised and maintained.Accordingly, this approach reduces or obviates a marked reduction in TCell activity in vitro which is observed in preparations of lymphocytesand APCs isolated using, for example, standard Ficoll gradients from awhole blood sample, when the blood sample is stored for several hoursprior to the isolation step.

In a preferred non-limiting embodiment, the present invention providesfor a method of detecting antigen-specific T cells in a subject,comprising (a) collecting a whole blood sample from the subject, (b)storing the sample for at least 6 hours after the collection, whereinstorage may optionally include or embody shipment or transport of thewhole blood sample, (c) purifying cells comprising lymphocytes (e.g., Tcells) and antigen presenting cells from the stored sample by a processselected from the group consisting of positive affinity selection andnegative affinity selection in order to remove granulocytes, (d)contacting the purified cells with a surface carrying an immobilizedantibody to a cytokine, (e) presenting to the purified cells anactivating amount of the antigen in the absence of any antigenpresenting cells pre-cultured with the antigen, (f) incubating thepurified cells under conditions to permit release of the cytokine butwhere the incubation time is not sufficient to effect differentiation ofprecursor effector T cells (or memory T cells) to immediate effector Tcells, and (g) detecting the cytokine released in response to theantigen. and bound to the immobilized antibody, wherein the whole bloodsample and the purified cells are not subject to conditions sufficientto effect differentiation of precursor effector T cells (or memory Tcells) to immediate effector T cells before the incubation.

In a further preferred non-limiting embodiment, the present inventionprovides for a method of detecting antigen-specific immediate effector Tcells in a subject, comprising (a) collecting a whole blood sample fromthe subject, (b) storing the sample for at least 6 hours after thecollection, wherein storage may optionally include or embody shipment ortransport of the whole blood sample, (c) purifying cells comprisinglymphocytes (e.g., T cells) and antigen presenting cells from the storedsample by a process selected from the group consisting of positiveaffinity selection and negative affinity selection in order to removegranulocytes, (d) contacting the purified cells with a surface carryingan immobilized antibody to a cytokine, (e) presenting to the purifiedcells an activating amount of the antigen in the absence of any antigenpresenting cells pre-cultured with the antigen, (f) incubating thepurified cells under conditions to permit release of the cytokine butwhere the incubation time is not sufficient to effect differentiation ofprecursor effector T cells (or memory T cells) to immediate effector Tcells, and (g) detecting the cytokine released in response to theantigen and bound to the immobilized antibody, wherein the whole bloodsample and the purified cells are not subject to conditions sufficientto effect differentiation of precursor effector T cells (or memory Tcells) to immediate effector T cells before the incubation.

The present invention also provides a kit for storing a whole bloodsample comprising a standard heparinized tube and cell growth medium.The invention also provides a kit for carrying out an ELISPOT assay oncells which have been stored and purified by a method as disclosedherein, wherein the kit comprises:

-   -   (i) a cytokine-specific antibody, optionally attached to a        microtiter plate, and    -   (ii) one or more of the ligands mentioned herein which can be        used in affinity selection, optionally bound to a solid support,        and    -   (iii) optionally instructions for carrying out the ELISPOT assay        and/or method of storing and purifying the cells.

Further, the invention also provides a kit for purifying a population oflymphocytes and APCs according to the invention, wherein the kitcomprises one or more of the ligands mentioned herein, optionally boundto a solid support. The kit may also comprise instructions for carryingout the method in accordance with the invention.

In other embodiments of the invention, the whole blood sample is treatedusing a positive or negative affinity selection step to isolate apopulation of T cells and APCs for use in the CMI assay, such as anELISPOT assay without storing the samples. In a further embodiment, thepositive or negative affinity selection methods may be used to isolate apreparation of cells for use in a CMI assay such as an ELISPOT assay,such cells being derived from whole blood samples which have beentreated by dilution and stored.

Any of the above embodiments may be carried out on a sample from ahuman, such as a human that is suspected of having a disease which canbe diagnosed or monitored by a CMI assay (for example, an ELISPOTassay).

The invention is hereinafter described in more detail by reference tothe following Examples.

Example 1 Treatment by Dilution with Culture Medium

Immediately after venipuncture, one 10 ml Lithium Heparin Vacutainer (BDBiosciences, Oxford, UK) of whole blood from each of a total of 78donors and was processed over the space of 6 months, using the T-SPOT.TBassay kit (Oxford Immunotec, Abingdon, UK). The method was carried outaccording to the manufacturer's instructions accompanying the kit. Thismethod comprises: a) a standard Ficoll method for preparation ofPeripheral Blood Mononuclear Cells (PBMCs)—see under Sample Collectionand Preparation, Procedure 2 “alternative blood collection methods” andNote 2.; b) viable cells were counted using the Trypan Blue DyeExclusion method (Freshney, R. (1987) Culture of Animal Cells: A Manualof Basic Technique, p. 117, Alan R. Liss, Inc., New York); c) cells werediluted in GIBCO AIM-V (Invitrogen, Paisley, UK) to give 250,000 cellsper 100 μl.; d) 250,000 (100 μl) of the Ficoll-extracted cells wereadded to each microtiter plate well pre-coated with anti-gammainterferon, together with one of the following: phytohaemagglutinin(PHA) positive control; or specific test antigens from M. tuberculosis(Panel A; Panel B); or from cytomegalovirus/epstein barr virus/influenzavirus (CEF; Mabtech, Sweden), which antigen is not provided in theT-SPOT.TB kit; e) the plate was incubated for 16 to 20 hours (37° C., 5%CO₂), washed four times with phosphate buffered saline (PBS) beforeadding an alkaline phosphatase-conjugated secondary antibody andincubating for 1 hour at 2-8° C.; f) the wells were washed four times,and 5-bromo-4-chloro-3′ indoylphosphate/Nitro Blue Tetrazolium(BCIP/NBT) then added and incubated for 7 minutes. After the spots haddeveloped, the reaction was stopped using distilled water; g) afterdrying at 37° C. for 4 hours, the number of spot forming cells (SFC) foreach well was recorded and analysed.

The remaining blood from each donor was diluted 1:1 in AIM-V and storedat room temperature (18-25° C.) in the dark overnight for 24 hours.

On Day 2, the T-SPOT.TB assay was performed on the stored bloodincluding the standard Ficoll method for preparation of PBMC. TheT-SPOT.TB assay was then conducted to determine the SFC counts, using apositive control (PHA) and a nil control (lacking antigen). The nilcontrol was subtracted from the test antigen well SFC count. If any ofthe antigen wells contained six or more spots and the nil controlcontains zero spots, then the sample was deemed ‘reactive’ for thatantigen under test. The Tables below show the relationship between freshand diluted and stored blood. Reactive and non-reactive results areshown using Panel A, Panel B and CEF wells for each donor blood sampleboth before and after overnight blood storage (following 1:1 dilution inAIM-V) at room temperature (RT). The percentage of the total number ofdonors is also indicated. CEF antigen was dissolved in AIM V, and waspresent in the assay at a level of 5 μg/ml.

Fresh Blood Diluted/Stored Blood Reactive Non-Reactive Panel A AntigenReactive 3 (3.8%) 1 (1.3%) Non-Reactive 2 (2.6%) 72 (92.3%) 96.2%clinical concordance Panel B Antigen Reactive 5 (6.7%) 0 (0%)  Non-Reactive 3 (4%)   67 (89.3%) 96% clinical concordance CEF AntigenReactive 28 (77.8%) 0 (0%)   Non-Reactive  5 (13.9%) 3 (8.3%)

Results demonstrate 86.1% clinical concordance (assuming that 6 spotsgreater than nil control indicates reactivity).

Thus, the addition of medium to the blood sample enables a positive SFCresponse to be measured in PBMC after 24 hours storage, a response thatis similar to that seen when measured on blood processed immediatelyafter collection. Blood collected and stored for 24 hours without theaddition of medium demonstrated a significant loss in the measurable CMIresponse (results not shown).

Example 2 Positive Affinity Selection

On Day 1, and within 2 hours of venipuncture, two 10 ml Lithium HeparinVacutainers of whole blood from each of nine donors were placed instorage, one in the dark at RT (18-25° C.) and the other in the fridge(2-8° C.).

At each time point, either 0-2 hours (fresh) or 24 hours (stored) postvenipuncture for each donor, 3 mls of whole blood were diluted 1:1 withwash buffer (PBS/0.1% BSA/2 mM EDTA) and mixed with 80 μl of a cocktailof specific magnetic Dynabeads, namely 111.45 (CD4); 111.47 (CD8);111.49 (CD14); and 111.43 (CD19), which were mixed in equal proportions(Invitrogen). These ‘beads’ positively select cells based on thepresence of specific differentiation (CD) markers: CD4 and CD8 for Tcell subsets; CD14 (monocytes) and CD19 (B cells and APC's). The sampleswere mixed at RT for 12 mins, and the beads were separated by placingsample tubes in a magnetic particle concentrator (MPC: Invitrogen,Paisley, UK) for 2 mins. The supernatant was discarded, and the beadswere washed twice in PBS and then separated again on the MPC. The beadswere then resuspended in 1 ml AIM-V and cells were counted using theTrypan Blue Dye Exclusion Method. Cells were diluted in AIM-V to give250,000 cells per 100 μl.

In parallel, at each time point, either 0-2 hours (fresh) or 24 hours(stored) post-venipuncture for each donor, 5 ml of whole blood wasprocessed immediately using the standard Ficoll method as indicated inExample 1.

All processed samples were then assayed using the T.SPOT.TB assay kit(see Example 1), using antigens CEF (5 μg/ml), or PPD (1 μg/ml), orPPD/CEF (1 μg/ml and 5 μg/ml respectively) in the assay wells, none ofwhich are provided in the T-SPOT.TB kit; PPD (Product Code RT23) wasobtained from Statens Serum Institute (Copenhagen, Denmark); for CEF,see Example 1. PPD and CEF were used to identify CD4+ and CD8+ T cellsrespectively.

The following Table, incorporating the average of the results for alldonors, displays the SFC counts measured by ELISPOT for cells selectedfrom fresh or stored blood using CD4, CD8, CD14 & CD19-conjugatedmagnetic beads compared to use of Ficoll only. The blood stored at RTfor 24 hours and selected using beads has produced comparable results tothe ‘fresh bead’ samples, and yield better results than when ‘Ficollonly’ samples are stored at RT, and both ‘Ficoll only’ and ‘beads’ arestored at 4° C.

Stored (24 Hours) Fresh Ficoll Ficoll Beads Antigen Ficoll Beads (4° C.)(22° C.) (4° C.) Beads (22° C.) PPD 100 78 15 41 53 70 CEF 100 194 13 45111 149 PPD/CEF 100 136 14 43 82 109 PPD 71.40 18.21 35.06 67.34 49.67CEF 109.20 18.51 37.24 87.54 109.40 PPD/CEF 81.80 1.52 2.85 40.89 56.25

The results were normalised, assuming that ‘fresh’ samples, processedwith Ficoll only, yield an SFC count of 100% in the ELISPOT assay. Thetop three rows show the mean SFC counts, and the bottom three rows showthe standard deviations.

Example 3 Negative Affinity Selection

Immediately after venipuncture, two 10 ml Lithium Heparin Vacutainers ofwhole blood were collected from each of 4 donors over the space of 1month.

For one sample from each donor, a negative selection step was undertakenusing MACSi anti-CD15 magnetic beads (Miltenyi Biotech, BergischGladbach, Germany). The beads were resuspended thoroughly before use toensure a homogeneous dispersion. 400 μl of beads were added to 4 ml ofwhole blood in a 15 ml conical tube. The tubes were incubated at RTusing the MACSimix (Miltenyi Biotech) tube rotator at medium speed (8rpm) for 15 minutes. The tubes were placed in the MACSiMag separator(Miltenyi Biotech), and the beads allowed to adhere to the wall of thetube for 2 minutes. Retaining the tube in the magnet, the supernatantswere removed using a pipette and retained in a fresh tube. The tubescontaining the supernatants were again placed in the MACSiMag separatorand any residual beads allowed to adhere to the wall of the tube for 2minutes. Retaining the tube in the magnet, the supernatant wastransferred to a 50 ml conical tube. 16 ml of freshly prepared 1× redblood cell (RBC) lysis buffer (prepared by dilution of 10× lysis buffer:1.55M NH₄Cl, 100 mM KHCO₃, 10 mM EDTA) were added and the tubesincubated at room temperature for 5 minutes. The samples werecentrifuged at 300×g for 10 minutes. The pelleted cells were washedtwice in lysis buffer (5 ml) and centrifuged each time at 300×g for 5minutes. The cells were washed once in RPMI and diluted in 1 ml of AIM-Vfor use in an ELISPOT assay.

In parallel with this method, and using a second sample from each donor,5 ml of whole blood was processed by the standard Ficoll method asindicated in Example 1.

Purified PBMC samples were then assayed in the T.SPOT.TB assay, asoutlined in Example 1, using CEF (5 μg/ml) as antigen for Donor 1,2 and4 samples; and Panel A and B antigens (33 μg/ml) for the Donor 3samples.

The SFC count obtained with PBMCs (shown in the Tables below) preparedusing a negative selection approach with MACSi anti-CD15 magnetic beadsdemonstrated high equivalence to those PBMCs that were prepared usingFicoll gradients. This antibody-based method of PBMC isolation proved tobe technically feasible and produced highly reproducible results in theT-SPOT.TB assay.

Donor 1 Donor 2 Donor 3 Donor 4 Ficoll MACSi Ficoll MACSi Ficoll MACSiFicoll MACSi CEF 240 239 100 111 − 0 0 − 0 0 248 235 98 124 + >100 >1000 0 Mean 244 237 99 117.5 panel A 41 31 + 116 100 Std dev 5.66 2.83 1.419.19 45 42 116 100 44 38 CEF 187 240 panel B 179 99 210 214 157 106 180189 151 83 Mean Mean − 0 0 Panel A 43.3 37 + 116 100 Panel B 162.3 96CEF 192.3 214.3 St Dev St Dev Panel A 2.08 5.57 − 0 0 Panel B 14.7411.79 + 0 0 CEF 15.70 25.50 (N.B. For Donors 3 and 4, (−) wells indicateabsence of antigen; (+) wells indicate presence of PHA (5 μg/ml).

Example 4 Negative affinity Selection

Two separate experiments were performed, each using pooled whole bloodfrom a group of either 15 or 11 donors.

For each experiment: a) on Day 1, immediately after venipuncture, one 10ml Lithium Heparin Vacutainer of whole blood from each donor pool wasprocessed according to the standard Ficoll method (see Example 1).Viable PBMC were counted using the Trypan Blue Dye Exclusion Method.Cells were diluted in cell culture medium (AIM-V) to give 250,000 cellsper 100 μl; b) the remaining blood was stored in the dark, undiluted,either in a fridge at 2-8° C. or at RT (18-25° C.) for 24 hours; c) onDay 2, stored blood samples were processed according to the standardFicoll method for PBMC preparation, and viable cells were counted anddiluted as described above (see paragraph 1, this Example).

In parallel samples processed in each experiment, both on Day 1 (fresh)and Day 2 (stored) for each donor, 4.5 mls of whole blood was added to15 ml centrifuge tubes, and 225 μl of RosetteSep (StemCell, Vancouver,BC, Canada) was then added to each tube to bind the RBC to thegranulocytes using a tetramer complex containing anti-CD66b andglycophorin A antibodies. Samples were mixed gently and left to incubatefor 20 minutes at RT. Samples were then diluted 1:1 with RPMI, and thePBMCs were isolated from the granulocyte-depleted cells by the standardFicoll method (see Example 1), thus allowing the RBC and granulocytes tobe separated from lymphocytes and APCs. The latter cells were thenwashed and processed according to the manufacturer's instructionsaccompanying the kit—see Example 1.

Results are shown below. In the two separate experiments performed, oneused CEF (5 μg/ml) as antigen, and the other used PPD (1 μg/ml) in theassay. Top Table: CEF-induced SFC counts mean of 15 donors) for Ficolland RosetteSep treatment at 4° C. and RT. Bottom Table: PPD-induced SFCcounts (mean of 11 donors) for Ficoll and RosetteSep treatment followingstorage at 4° C. and RT. The results demonstrate that samples stored at4° C. and then subjected to anti-granulocyte antibody treatment followedby Ficoll purification of PBMC are equivalent to standard ‘fresh’samples, and also yield higher SFC counts than when samples are storedat RT prior to anti-granulocyte antibody treatment.

Fresh Ficoll, RosetteSep, RosetteSep, Ficoll 4° C. 4° C. Ficoll, RT RTCEF Mean 80 54 78 39 60 sum 4870 3252 4759 2388 3681 % diff from −33.2−2.3 −51 −24.4 fresh Ficoll n = 15 donors, 61 replicates PPD Mean 22.69.4 21.4 8 17.4 sum 722 301 685 256 558 % diff from −58.31 −5.12 −64.54−22.71 fresh Ficoll n = 11 donors, 32 replicates

Example 5 Filtration

The feasibility of using back flushed leukocyte filter membranes (PALLMedical) and also the stability of the cells captured on such membraneswas investigated at T0 and T24 hours post venipuncture. The capturedcells were stored on the membranes in a fridge (2-8° C.) from one donorand RT (18-25° C.) for the second donor. This membrane separation methodwas performed along side a standard Ficoll PBMC separation method ateach time point and temperature.

The membranes that were required to be stored followed steps 1-3 (seebelow) and then the appropriate storage condition and duration subjectedon them. The membrane holder devices were then removed from the storagearea and subjected to steps 4-7 (see below) at the relevant time point.The Ficoll control storage samples were kept in the dark at RT and werediluted 1:1 in AIMV cell culture media until required for processing viathe standard Ficoll separation method. The membrane procedure isdescribed below:

Steps 1-3 consisted of; 10 ml of WASH BUFFER 1 being filtered throughthe membrane, using the transfer syringe, into the waste bottle. 10 mlof whole blood sample together with 50 ml of WASH BUFFER 1 was mixedfirst and then filtered through the LK4 membrane, using the transfersyringe, in to a waste bottle. 50 ml of cooled WASH BUFFER 1 wasfiltered through the LK4 membrane, using the transfer syringe, into thewaste bottle.

Steps 4-7 consisted of: the filter membrane being inverted eitherimmediately or post 24 h (depending on time point). 50 ml of cooledREMOVING BUFFER 1 was filtered back through the membrane, using the backflush syringe, into a 50 ml falcon tube. The cell suspension was spun at2000 rpm for 5 mins at RT and then re-suspended in 10 mls of AIMV. Thecells suspension was again spun at 2000 rpm for 5 mins at RT and thenre-suspended in 1 ml of AIMV media and counted according to the standardFicoll separation method. (WASH BUFFER 1: 500 ml AIMV media bufferedwith 20 mM HEPES and 1% [w/v] Dextran T40) (REMOVING BUFFER 1: 500 mlAIMV media buffered with 20 mM HEPES and 0.1% [w/v] Dextran T40).

The T-SPOT.TB assay was then performed as indicated in Example 1 andalso in the manufactures guidelines accompanying every kit.

The following table describes a summary of results from the T-SPOT.TBtests carried out for this investigation. A comparison of the CEF spotcounts with regard to time and temperature of the membrane experimentsis shown. This was carried out in terms of the membranes performance inrelation to the relevant Ficoll controls carried out on the same day andalso their relation at T0.

CEF Percentage of Count Percentage of Ficoll control at Date DonorMethod Temp (ave, n = 4) Ficoll Control T0 T0 Apr. 05, 2006 0002 Ficoll(RT) 45.5 ~ ~ LK4 2-8° C. 11 24.2 ~ 0008 Ficoll (RT) 44.4 ~ ~ LK4 RT21.25 47.9 ~ T24 May 04, 2006 0002 Ficoll (RT) 47.5 ~ 104.4 LK4 2-8° C.10.5 22.1 23.1 0008 Ficoll (RT) 33.75 ~ 76.0 LK4 RT 4.75 14.1 10.7

It can be seen from the above table, the membranes used with fresh wholeblood, stored at 2-8° C. and 18-25° C. (RT) at both T0 and T24 haveprovided PBMCs which produce sufficient CEF signal for the use in theELISPOT assay. The concept of using back flushed filter membranes toharvest PBMCs capable of eliciting a response in the ELISPOT assay hasbeen proven in this case.

Example 6 Storage Time Before Negative Affinity Selection

Two separate experiments were performed, using samples of whole bloodfrom each of 15 donors.

For each experiment: a) on Day 1, immediately after venipuncture, one 10ml Lithium Heparin Vacutainer of whole blood from each donor wasprocessed (‘fresh’ samples), according to the standard Ficoll method forPBMC preparation; b) the remaining blood from each donor was stored inthe dark, undiluted, in a fridge at 2-8° C. (stored samples) for varioustimes (16 to 72 hours); c) on Day 2, the stored samples were incubatedwith RosetteSep (StemCell, Vancouver, BC, Canada) as described inExample 4, then processed according to the standard Ficoll method forPBMC preparation. Counting and dilution of PBMC was then carried out,and samples processed, according to Example 4, except that in the twoseparate experiments performed, one used CEF (5 μg/ml) as antigen, andthe other used CEF/PPD (5 μg/ml and 1 μg/ml respectively) in the assay.

The results shown below represent the cumulative SFC counts recorded forall individual donor samples, after each had been subjected to varyingprocessing conditions, using either CEF antigen (n=10) or using both CEFand PPD antigens (n=5), based on summing the mean of the result of threeseparate ELISPOT assays for each processed sample.

The data demonstrate that samples stored for between 16 and 24 hours at2-8° C. can subsequently be ‘stabilised’ with anti-granulocyte antibodyprior to being processed with Ficoll, whereas longer storage timesfollowed by the same purification conditions give rise to a gradualreduction in SFC counts in the ELISPOT assay.

Fresh RosetteSep, Stored at 2-8° C. Antigen Ficoll 16 hrs 20 hrs 24 hrs48 hrs 72 hrs CEF 940 900 846 892 661 354 CEF/PPD 481 502 476 584 324108

Similar results are obtained when samples are stored at RT for up to 48hours prior to being ‘stabilised’ and processed with Ficoll.

Example 7 Negative Affinity Selection using Anti-CD 15 Magnetic Beads

Two separate experiments were performed, using samples of whole bloodfrom each of 8 donors. The blood was collected into 6 ml Lithium HeparinVacutainers. Cells were separated from whole blood using either (i)Negative Affinity Selection or (ii) Ficoll.

(i) Negative Affinity Selection. 4 ml of blood from each donor wasprocessed immediately after collection (‘fresh’ samples) using theNegative Affinity Selection procedure described below; the remainder wasstored at RT, and 4 ml of blood from each donor processed after either24 hours or 48 hours of storage.

At each time point 4 ml of whole blood was added to 36 ml cold AmmoniumChloride Lysis Solution (8.26 g/L Ammonium Chloride, 1.0 g/L Potassiumbicarbonate, 0.037 g/L EDTA, pH 7.2-7.6), mixed and incubated at 2°-8°C. for 15 minutes. The samples were centrifuged at 300×g for 5 minutes,the supernatant removed, and the cells re-suspended in 10 mls washbuffer (PBS, 2 mM EDTA, 0.5% BSA). The samples were centrifuged again at300×g for 5 minutes, and the supernatant removed. The cells werere-suspended in 80 μl wash buffer. 20 μl of CD15 MicroBeads (MiltenyiBiotech) to label granulocytes, and 20 μl CD235a MicroBeads (MiltenyiBiotech) to label residual RBC, were added to each sample. Samples weremixed and incubated for 15 minutes at 2°-8° C. 2 mls wash buffer wasadded, and samples centrifuged for 5 minutes at 300×g. The supernatantswere removed and each cell pellet was re-suspended in 500 μl of washbuffer. These labelled samples were then depleted using LD MACS cellseparation columns according to the manufacturer's instructions(Miltenyi Biotech). The unlabelled cells were collected from thecolumns, centrifuged to pellet, and re-suspended in 1 ml AIM V medium.

(ii) Ficoll. In parallel, using the standard Ficoll method (see Example1), 4 ml of blood from each donor was processed immediately aftercollection (‘fresh’ samples); the remainder was stored at RT, and 4 mlof blood from each donor was processed after either 24 hours or 48 hoursof storage. Cells collected were re-suspended in 1 ml AIM-V medium.

At each time point, cells collected from each of the cell isolationprocedures were counted and diluted in AIM-V to give 250,000 cells/100μl. All samples were then assayed in the T-SPOT.TB assay as outlined inExample 1 using CEF (Mabtech, 0.66 μg/ml) as antigen. FIG. 1 below showsthe data obtained from 4 representative donors of the 8 donors tested.

The data shows that the blood samples processed with Negative AffinitySelection, using anti-CD15, after 48 hours storage gives similar spotcounts to samples prepared using Ficoll with blood processed within 8hours of collection.

1. A method of performing a cell-mediated immunoassay, comprising:collecting a whole blood sample from said subject; (ii) maintaining saidsample for at least 6 hours after said collection, (iii) purifying apopulation of cells comprising lymphocytes and antigen presenting cellsfrom said maintained sample by a process comprising a positive ornegative affinity selection step in order to remove granulocytes; and(iv) using said population of cells in a cell-mediated immunoassay. 2.The method of claim 1, wherein said sample is maintained for at least 10hours.
 3. The method of claim 2, wherein said sample is maintained forat least 12 hours.
 4. The method of claim 3, wherein said sample ismaintained between 12 and 36 hours, 12 to 48 hours, or 24 to 48 hours.5. The method of claim 1, wherein said sample is maintained between 2-8°C.
 6. The method of claim 1, wherein said sample is maintained between18-25° C.
 7. The method of claim 1, wherein said cell-mediatedimmunoassay measures antigen-specific cytokine responses in immediateeffector T cells.
 8. The method of claim 1, wherein said processcomprises a negative affinity selection step wherein said maintainedsample is contacted with an antibody preparation comprising anti-CD66band glycophorin A antibodies to aggregate red blood cells andgranulocytes, and said aggregated red blood cells and granulocytes areremoved from said maintained sample by centrifugation.
 9. The method ofclaim 1, wherein granulocytes are removed in said process by contactingsaid maintained sample with a solid support comprising an anti-CD15ligand to remove CD15+ cells from said maintained sample.
 10. The methodof claim 9, wherein said solid support further comprises magnetic beads,and wherein said process further comprises contacting said maintainedsample with said beads to bind CD15+ cells to said beads, and separatingsaid beads having said CD15+ cells bound thereto from said maintainedsample.
 11. The method of claim 9, wherein red blood cells in saidmaintained sample are removed by lysis or filtration, prior to step(iv).
 12. The method of claim 1, wherein said purification leads to anincrease in the sample of both the ratio of T cells to total cells andthe ratio of antigen presenting cells to total cells.
 13. The method ofclaim 1, wherein said sample is not frozen before said purification. 14.The method of claim 1, wherein said purified cells are not frozen beforesaid use.
 15. The method of claim 1, wherein said process comprises apositive affinity selection step.
 16. The method of claim 15, whereinsaid positive affinity selection step selects all types of T cells. 17.The method of claim 15, wherein said process comprises contacting saidmaintained sample with a solid support having attached thereto ligands,wherein said ligands bind to cell surface proteins present on thesurface of said lymphocytes and antigen presenting cells, and whereinsaid lymphocytes and antigen presenting cells are bound to said solidsupport via said ligands.
 18. The method of claim 17, wherein said solidsupport comprises one or more ligands selected from anti-CD4, anti-CD8,anti-CD19 and anti-CD14 ligands to purify CD4+, CD8+, CD19+ or CD14+cells from said maintained sample.
 19. The method of claim 17, whereinsaid bound lymphocytes and antigen presenting cells are separated fromsaid solid support before said use in step (iv).
 20. The method of claim17, wherein said bound lymphocytes and antigen presenting cells areretained on said solid support before said use in step (iv).
 21. Themethod of claim 17, wherein said solid support comprises magnetic beads.22. The method of claim 21, wherein each said magnetic bead has boundthereto anti-CD4, anti-CD8 and anti-CD 19 ligands.
 23. The method ofclaim 21, wherein each magnetic bead has bound thereto one or moreligands selected from anti-CD4, anti-CD8 and anti-CD19 ligands, andwherein said beads are mixed together to provide said solid support forbinding to CD4+, CD8+ and CD19+ cells.
 24. The method of claim 15,wherein red blood cells in said maintained sample are removed by lysisor filtration, prior to step (iv).
 25. The method of claim 1, whereinsaid cell-mediated immunoassay is an ELISPOT assay.
 26. The method ofclaim 1, wherein said subject is a human.
 27. The method of claim 1,wherein the step of maintaining the whole blood sample includes shippingor transporting said sample from one geographical location to another.28. The method of claim 26, wherein said cell-mediated immunoassay is todiagnose or monitor a disease.